4-(Indol-3-ylmethyl)-4-hydroxy-glutamic acid (3-(1-amino-1,3-dicarboxy-3-hydroxy-butane-4-yl)-indole) (hereinbelow referred to as “monatin”) represented by the following structural formula (3) is present in roots of a plant Schlerochitom ilicifolius and is a particularly promising low-calorie sweetener because of its remarkably high sweetness intensity (JP 64-25757 A):
4-(Indol-3-ylmethyl)-4-hydroxy-glutamic acid
Monatin has two asymmetries at positions 2 and 4, and the natural stereoisomer has been reported to be a (2S,4S) isomer. Other stereoisomers have been synthetically prepared, and three stereoisomers have been identified. It has been confirmed that any of them has sweetness intensity that is several ten to several thousand times higher than that of sucrose (Table 1).
TABLE 1Sweetness of monatin isomersStereoisomerSweetness (vs. sucrose)2R, 4R2700 times2R, 4S1300 times2S, 4R 300 times2S, 4S 50 times
As is shown in Table 1, not only naturally occurring (2S,4S)-monatin but also all other stereoisomers have the sweetness intensity with high scale factor. Particularly, (2R,4R)-monatin has an remarkably high sweetness intensity which is 2,700 times higher than that of sucrose and is the most highly expected as a sweetening agent or a sweetening agent ingredient (sweetener). Therefore, it has been desired to develop a method for efficiency producing monatin with high content of (2R,4R)-monatin.
Five examples of monatin production processes have been reported. Details thereof are described in (1) and (3) to (6) of the following prior art references.
(1) U.S. Pat. No. 5,994,559
(2) European Patent Publication No. 0736604 A
(3) Tetrahedron Letters, Vol. 42, No. 39, pages 6793-6796, 2001
(4) Organic Letters, Vol. 2, No. 19, pages 2967-2970, 2000
(5) Synthetic Communication, Vol. 24, No. 22, pages 3197-3211, 1994
(6) Synthetic Communication, Vol. 23, No. 18, pages 2511-2526, 1993
(7) Taylor et al., Journal of Bacteriology, Vol. 180, No. 16, pages 4319, 1998
However, none of the aforementioned references refers to any stereoselective method for producing (2R,4R)-monatin. In addition, all of the disclosed methods require multiple steps, which impedes practical production on an industrial scale.
In such a situation, the present inventors have proposed a novel method for producing monatin from 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid (hereinbelow referred to as IHOG) using an enzymatic reaction shown in the following formula (4).

This novel method utilizes an enzyme which catalyzes an amination reaction at position 2 of a monatin precursor (IHOG), to thereby produce monatin from IHOG. Aminotransferase is one of enzymes which catalyze the amination reaction of IHOG. Employment of the D-aminotransferase results in selective production of 2R-monatin, and employment of a L-aminotransferase results in selective production of 2S-monatin. That is, employment of the D-aminotransferase as the enzyme for catalyzing the reaction results in selective production of the 2R isomer, i.e. the highly sweet isomer, as a result of transfer of an amino group from a D-amino acid as an amino donor to position 2 of IHOG.
Studies by the present inventors have revealed that the D-aminotransferase which catalyzes a reaction of the substrate IHOG to produce 2R-monatin is present in microorganisms belonging to genus Bacillus or Paenibacillus. However, even when using the D-aminotransferase derived from these microorganism, it has been difficult to efficiently produce monatin containing (2R,4R)-monatin at a high ratio.
One of the reasons for such an inefficient (2R,4R)-monatin production appears to be poor recognition of the asymmetry at position 4 of IHOG by the D-aminotransferase derived from these microorganisms. That is, when the D-aminotransferase derived from the microorganisms belonging to genus Bacillus or Paenibacillus is allowed to act upon a racemic mixture as to position 4 (sometimes abbreviated hereinbelow as 4R, S-IHOG), it acts upon both 4R-IHOG and 4S-IHOG and produces both (2R,4R)-monatin and (2R,4S)-monatin at an almost equal ratio. Thus, even when using the D-aminotransferase derived from these microorganisms, it has been impossible to produce monatin having an optical activity at position 4.
Another reason therefor would be instability of IHOG, the material for producing monatin, in a certain pH. In order to test stability of IHOG in the amination reaction of IHOG, the present inventors measured a change of IHOG concentration with time in an amination reaction solution of IHOG with no microbial cell addition. The reaction was performed by shaking a test tube containing 1 ml of reaction solution composed of 100 mM potassium phosphate buffer (pH 8.3), 300 mM 4R, S-IHOG, 600 mM DL-Ala and 1 mM pyridoxal-5′-phosphate at 37° C. for 40 hours. As a result, a residual ratio of 4R, S-IHOG was decreased to 81% after 16 hours, 70% after 24 hours and 57% after 40 hours, respectively. It was found that IHOG was decomposed with the lapse of time. It is presumed that this phenomenon is due to a decomposition reaction where IHOG is decomposed into 3-indole-pyruvic acid and pyruvic acid and a cyclization reaction of IHOG, to consume IHOG in the reaction solution before being converted to monatin. That is, the reaction catalyzed by the D-aminotransferase derived from the genus Bacillus or Paenibacillus is not sufficiently fast, and a part of IHOG becomes unavailable for the amination due to the decomposition and the cyclization before the amination. This is thought to be one reason why (2R,4R) monatin can not be efficiently produced.
Accordingly, it has been desired to develop a method for efficiently producing (2R,4R)-monatin which has the highest sweetness intensity among the monatin isomers.
A task to be accomplished by the present invention is to provide a D-aminotransferase capable of efficiently producing the (2R,4R) isomer of the glutamic acid derivatives such as monatin and analogues thereof.